Project Summary Pre-sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are essential drivers of physiological and pathological increases of sympathetic nerve activity (SNA). Perhaps their most robust property is their resting state of discharge quiescence. Early studies linked quiescence to the dominance of synaptic inhibition, but mechanisms that establish and defend GABAergic inhibitory tonus in the PVN are understood only on a rudimentary level. This is an important knowledge gap because pathogenic factors that increase PVN-driven SNA must ultimately subvert or overwhelm mechanisms that regulate the quiescent resting state of PSNs. In preliminary studies, we uncovered a presynaptic mechanism that is novel to the PVN, referred to as ?Glutamate-GABA strengthening (GGS)?, that increases GABAergic inhibition in pace with synaptic glutamate (Glu) spillover. To do so, GGS regulates the amplitude of GABA-A receptor-mediated inhibitory postsynaptic currents (IPSCs) through uptake of synaptically released Glu, ostensibly into local GABA terminals, by the neuronal excitatory amino acid transporter 3 (EAAT3). Once internalized, Glu is converted to GABA and GABA molecules are packaged into synaptic vesicles at greater than normal density. Stressors that acutely increase PVN-driven SNA are hypothesized to increase synaptic Glu release without changing extrinsic GABAergic input. As a result, ?over-filled? GABA vesicles are released that dampen excitation and aid restoration of PSN quiescence. During chronic sympathoexcitation challenges accompanied by reduced GABA input, GGS is subverted (due to low GABA release) and can therefore provide little opposition to synaptic excitation. Proposed studies will use state-of-the-art transgenic mouse models, optogenetics and virus-mediated gene over-expression and CRISPR-Cas9 knockdown to assess mechanisms and functional outcomes of GGS. Kinetics, sensitivity and efficacy of GGS will be established at the single PSN level using a novel horizontal brain slice preparation that preserves Glu input from the forebrain median preoptic nucleus (MnPO) as well as GABA input from the PVN peri-nuclear zone (PNZ). Retrogradely transported AAV will be injected into the PVN of vGlut2-Cre mice to express channelrhodopsin (ChR2) in glutamatergic MnPO-PVN neurons. Optogenetic activation will determine the capacity of MnPO inputs to drive GGS amongst RVLM-projecting PVN PSNs. Using vGlut2fl/fl mice, we will determine functional effects of GGS on GABA-A receptor inhibitory tone and SNA responses to forebrain angiotensin II (AngII) and hyperosmolality when glutamatergic MnPO neurons have normal (vGlut2 intact) or diminished (vGlut2 knockdown) capacity to release Glu from PVN synapses. To further illuminate in vivo mechanisms and efficacy of GGS, EAAT3 on PNZ GABA inputs to the PVN will be increased and decreased to grade PVN GABAergic tonus and the magnitude of PVN-driven SNA responses to (1) acute forebrain AngII and hyperosmolality as well as (2) sub-acute water deprivation and high salt intake. Proposed studies will provide unprecedented mechanistic insight into the physiological role GGS plays in generating and defending PVN PSN quiescence, and are essential for advancing the goal of preventing and reversing disease-promoting sympathoexcitation.